Mercaptopyruvate added to activated PFL generates two sulfur based radicals thereby inactivating the enzyme. For one of the radicals, hyperfine coupling to a single ~-methylene hydrogen of a disulfide radical is indicated. Deuteration of cys 418 and 419, residues required for catalysis, changes the EPR consistent with the radical being formed on a cysteine residue of the protein. Mutant enzymes C418S and C419S do not form the radical species observed.